The invention relates in general to microchip laboratory systems which serve to carry out chemical, chemical-physical, physical, biochemical and/or biological processes, in particular for the analysis or synthesis of substances on a carrier which features a microfluidic structure. The essentially flat carrier in this situation features a micro-channel structure, by means of which the substances are capable of movement in accordance with the channel structure under the imposition of a potential particularly an electrical potential. In particular, the invention relates to such microchip systems in which a micro spray tip is provided for spraying substances to the outside particularly for the insertion spraying of substances into a mass spectrometer. In addition, the invention relates to a process for the manufacture of a microchip featuring such a micro spray tip, as well as a device for handling such a microchip.
The rapid progress in the sector in question can best be illustrated by way of the corresponding developments in the microelectronics sector. In the chemical analysis sector, too, not least with regard to clinical outpatient diagnosis, there is a substantial demand for existing stationary laboratory equipment to be integrated into portable systems, or for such systems to be miniaturised accordingly. An overview of the latest developments in the sector of laboratory microchip technology can be found in a collection of pertinent specialist publications edited by A. van den Berg and P. Bergveld under the title xe2x80x9cMicrototal Analysis Systemsxe2x80x9d, published by Kiuwer, Academic Publishers, Netherlands, 1995. The starting point for these developments was the already established method of what is referred to as xe2x80x9ccapillary electrophoresisxe2x80x9d, with which efforts have already been made in the past to implement the system on a planary glass microstructure.
FIG. 1 shows a conventional laboratory microchip. As shown on the upper surface of a substrate or carrier 10, microfluidic structures are applied, which serve to accommodate and transport substances. The carrier 10 may be made, for example, of glass or silicon, whereby the structures can be created by means of a chemical or laser-supported etching process. To accommodate a substance which is to be examined (referred to hereinafter as the xe2x80x9csubstance specimenxe2x80x9d) on the microchip, one or more indentations 11 are provided on the carrier, which serve as a reservoir for the individual substance specimen. For the purpose of conducting the experiment, the substance specimen is initially moved along a transport channel 15 on the microchip. In the present embodiment, the transport channel is formed by a V-shaped groove. There are, however, in principle other embodiments of the transport channel possible, such as rectangular or circular profiled cut-outs or grooves. Other depressions 12, serving likewise as substance reservoirs or wells, accommodate the reagents required for the performance of the experiment. In the present example, this involves two different substances, these being initially conducted by means of corresponding transport channels 16 to a point of intersection 17, where they intermix and, after chemical analysis or synthesis has been carried out if appropriate, they form the product which is ultimately to be used. At a further point of intersection 18, this reagent then encounters the substance specimen which is to be examined, whereby both substances are likewise intermixed.
The substance which is formed in this overall manner then runs through a meander-shaped transport section 19, which serves essentially to enlarge artificially the lengths of the distances available for the reaction between the substance specimen and the reagent. In the present example, in a further indentation 13, formed as a substance reservoir or well, an additional reagent is contained which is conducted to the substance mixture which already pertains, at a further point of intersection 21.
In the present example, the substance reaction which is actually to be examined takes place immediately adjacent to the point of intersection in reference 21. The detection of this substance reaction then takes place within a measurement field or area 22 of the transport channel by means of a detector, not shown here, for preference free of contact. An appropriate detector may in this case be arranged above or below the area 22. Once the substance has run through said area 22, it is conducted to a further indentation 14, which represents a substance sink for the substance waste residues formed overall during the reaction.
Finally, depressions 23 are provided on the microchip which function as contact surfaces for the application of electrodes, and which in turn allow for the imposition on the chip of the electrical voltages, and high voltages in particular, which are required for the operation of the chip. As an alternative, the contact for the microchip can also be provided by the introduction of an appropriate electrode to directly into the depressions 11, 12, 13, 14 provided for the accommodation of the substances. By means of a suitable arrangement of the electrodes 23 along the transport channels 15, 16, 19, 20 and a corresponding temporal and/or strength concordance of the fields used, a situation can now be attained in which the movement of the individual substances is effected in accordance with a temporal and volume profile which can be precisely predetermined, with the result that the kinetics of the reaction process taken as the basis in each case can be most precisely taken into consideration, and can be maintained respectively.
In the case of the movement of the substances by means of gas pressure (not shown here) within the microfluidic structure, it is necessary for the transport channels to be designed as conduits endosed all round, for example as hollow channels with predetermined cross-sections. In such an embodiment, it is therefore necessary for the depressions 23 to be designed in such a way that suitable pressure supply lines engage in them, duly sealed, in order for a pressure medium, such as a noble gas, to be introduced into the transport channels.
Miniaturisation of the microchips also allows for a substantial shortening of the transport paths for the substances, especially between the introduction point for the substances and the individual detection point for the measurement of a chemical reaction which is to be effected (see FIG. 1). From the sector of liquid chromatography and electrophoresis the principle is further known of a substance separation being implemented more rapidly in such systems, and therefore of the results of experiments likewise being provided more rapidly, and for the individual components to be separated with higher resolution than is possible in conventional systems. In addition to this, microminiaturised laboratory systems also allow for a substantial reduction in the consumption of substances, in particular of reagents, as well as a substantially more efficient mixing of the substance components.
A laboratory microchip of the type shown in FIG. 1 has been described, for example, in U.S. Pat. No. 5,858,195. The movement of the substances in the channels integrated on the microchip is controlled by means of electric fields, which are imposed along the transport channels. Because of the highly precise control of the substance movement which is achieved by this, and the very precise metering ability of the substance masses moved in each case, the substances can be mixed or separated precisely in relation to the desired stoichiometry, or physical-chemical reactions can be induced. The movement of the substances is effected in this case on the basis of what is referred to as electro-osmosis; i.e. the movement of individual substances within a substance mixture incurred by an electrical potential gradient. Substances move in electrical fields on the one hand due to their space charge. The space charge can for example be controlled by an appropriate chemically acidic environment. This is referred to as the electrophoretic flow. At the same time, each surface has a surface charge. Formed directly on the inner surface of a capillary, as a result of this surface charge (mostly negative) is a thin layer of the corresponding counter-charge (mostly positive) in the mobile phase (liquid). In the electrical field this thin layer migrates backwards and forwards, and takes the liquid in the interior of the capillaries with it. This flow is referred to as the electro-osmotic flow (EOF). The total flow is the sum of the EOF and the electrophoretic flow, whereby neutral molecules migrate with the EOF.
In particular, the microchip described in the aforesaid US Patent features a carrier with one or more points of intersection between the transport channels, at which intermixing of substances takes place. By the simultaneous application of different electrical potentials at different substance reservoirs or wells, the possibility is provided of the volume flows of the different substances being selectively controlled through one or more points of intersection, and therefore of a precise stoichiometric predetermination being rendered possible solely on the basis of the electrical potentials applied.
To detect the substance reactions taking place during an experimental procedure, optical procedures are mainly used, e.g. by the measurement of an absorption spectrum or fluorescence spectrum of the individual substance in each case. Optical detection in this situation requires transparent materials within the channel structure of such a microchip, such as glass or polymethyl metacrylate (PMMA). At the same time, it is necessary for the substance specimen which is to be detected to be marked in an absorbent manner either in the area of the individual wavelength of the measuring beam, or appropriately marked with fluorescing colouring agents. The restrictions which this induces in the selection of the substance specimens imply considerable disadvantages for such microfluidic measuring devices.
In addition to this, in many applications, such as in the field of protein analytics, optical detection is often difficult or can only be effected with considerable technical effort. In the prior art it was therefore proposed that a mass spectrometer (MS) be connected to a microfluidic microchip of the type described in the preamble. Usually an arrangement referred to as an xe2x80x9celectrospray interfacexe2x80x9d (ESI) is used in this situation, in order for the liquid substance specimens present for the MS detection to be ionised beforehand. xe2x80x9cElectrosprayxe2x80x9d ionisation serves in this case to generate ions for the mass-spectroscopic analysis of chemical or biological substance specimens. An ESI pertains when a liquid in a capillary tip (spray tip) is subjected to an electrical potential of a value of some 1-4 kV (kiloVolt). The high electrical field induces charges on the surface of the liquid in the area of the spray tip. The spraying off or dispersant spraying of the substance in the area of the spray tip occurs as soon as the Coulcombe forces are great enough to overcome the surface tension forces present in the liquid. lonisation by means of an ESI for flow rates such as typically arise in microfluidic structures (100-500 nl/min) do however require very high electrical field strengths, such as can only be created with very fine spray tips with a diameter of about 10-100 xcexcm (micrometres).
Pertinent microfluidic systems of the type in question have been disclosed, for example, in the patent specifications WO 97/04297, WO 98/35376, and U.S. Pat. No. 5,788,166. The microfluidic system disclosed in WO 97/04297 features a micro spray tip which serves as an xe2x80x9cESIxe2x80x9d interface for the transfer of substance specimens to a mass spectrometer. In an embodiment of the type disclosed there, the spray tip is manufactured in a construction unit with one or more channels. In addition, the spray tip projects, in relation to the surface plane of the microchip carrier, vertically out of this plane, whereby the tip according to FIG. 2b is likewise manufactured in a construction unit with a cover plate. The actual channel system is created on the side of the carrier turned away from the spray tip, by the provision of an appropriately dimensioned cover plate.
In the printed specification referred to, a spray tip is described which is micro-mechanically manufactured from silicon. This spray tip is in this case formed from a supporting substrate, for preference a silicon carrier. The liquid substance specimen in this situation flows first into a channel structure as previously described. At one end of this channel structure a spray tip is created, in the form of a channel, which is in a substance conductive connection with the channel structure. The manufacture of the spray tip is effected by means of numerous manufacturing stages, whereby multi-stage depositing processes are encompassed to form what is referred to as a xe2x80x9csandwichxe2x80x9d. This sandwich features on the outer sides two silicon-nitrite layers in each case. The spray tip itself is formed in this case by means of an etching process. The silicon-nitrite layers in this case are initially deposited on a silicon substrate. Thereafter the silicon-nitrite is structured by means of a plasma, with the formation of the spray tip.
The ESI source described in U.S. Pat. No. 5,788,166 also features a micro spray tip of the type described in the preamble, and is particularly well-suited for the atomization of liquid substances for use in the sector of capillary electrophoresis with ultra-low flow rates. The spray tip is created by tensile extension (drawing out) of a heated quartz tube. The quartz tube in this case is processed by chemical etching and subsequent surface metal coating subsequent to the tensile extension. The extension of the tube leads in particular to the formation of a channel or capillary, slowly tapering to a point, located within a needle-shaped extension, which, in addition, runs into a tip with an extremely small internal diameter. The etching process leads to a further thinning of the outer wall of the needle, and therefore to a further reduction of the diameter of the tip. After the application of a metallised electrical contact on the outer wall of the needle, an electrically insulating cover layer is then applied, which contributes towards increasing the service life of the needle.
A spray tip drawn out of a glass capillary is also described in Anal. Chem. 98, 70, 3728-3734, whereby the glass capillary is adhesively bonded into a microchip after being drawn out. The electrical connection in this case is created either via a metallized tip or by means of what is referred to as a xe2x80x9cliquid junctionxe2x80x9d on the chip. A liquid junction of this type, as an electrical connection for the ESI is also described in Anal. Chem. 97/69, 1174-1178. Instead of a spray tip, however, in this case what is referred to as a spray cone (Tayler Cone) is provided directly on the surface of a microchip. The liquid junction in this case is formed as an additional channel shortly before the spray cone. In order to prevent the possibility of the specimen molecules not following the electrical field along these connection channels, the connection channel is coated in such a way that no field edge pertains within the connection channel which would cause an electro-osmotic flow (EOF) of the substances.
The generation of such micro-tips can also be effected in a known manner by lithographic deposit processes, for example by the formation of parallels on the surface of a microstructure, making use of what is referred to as a sacrificial layer, which is subsequently dissolved, and so forms a channel. As in the process proposed in WO 98/35376, the individual manufacturing stages in this case are:
1. Layer of silicon nitrite on silicon (as base)
2. Phosphosilicate glass (as sacrificial layer)
3. Layer of silicon-nitrite (as cover and side wall)
4. Free etching of the tip (silicon carrier is freely etched)
5. Sacrificial layer is etched away.
The principle is also known of manufacturing a micro-tip by deep etching or ionic etching of silicon structures in such a way that, after the etching, the micro-tip remains. Such a manufacturing process is disclosed, for example, in a contribution by R. E. Swenson at an IBC Conference held on Sep. 9, 1999.
The microtips described heretofore now have the disadvantage that, because of the relatively elaborate manufacturing processes involved, there are at present no microstructure couplings of laboratory microchips of the previously described type, such as mass spectrometers or the like, which are capable of commercialisation.
The object underlying the invention is therefore to provide a microchip which features a micro spray tip of the type described in the preamble, the manufacture of which is simplified in comparison with the prior art. The microstructure to be created in this situation is intended, in addition to simplified means of manufacture, in particular to allow for the most reliable means of manufacture of extremely fine spray tips with an outer diameter of 10-100 xcexcm (micrometres). A spray tip of this kind should also provide a hydraulic connection, created in the simplest manner possible, to the separation channel system provided in each case on the microchip, as well as a simplified electrical connection for the electrical field required for the dispersion spraying of the substances from the spray tip.
The object underlying the invention is also to provide a device for the handling of a laboratory microchip featuring a micro spray tip, which will facilitate or render easy the use of such a microchip, in particular the coupling of the microchip to a mass spectrometer.
The objects indicated are achieved according to the invention by the features of the independent claims. Preferred and advantageous embodiments of the invention are described in the dependent claims.
The special feature of the present invention in comparison with the prior art described in the preamble lies in the fact that the micro spray tip is prepared from a carrier material in such a way that the spray tip and the carrier are formed in a single piece, in particular in a monolithic manner. In addition to this, the channel structure provided on the microchip is arranged in the side of the carrier which is backwards to the spray tip, and can be automatically connected in a substance-conductive manner by means of the manufacturing method to be described in detail hereinafter.
The advantages of the proposed arrangement of the micro tip and of the manufacturing process relating to this lie in particular in the fact that the manufacture of the spray tip is rendered substantially easier, and that therefore a reduction in costs can be achieved in the manufacture of these tips, which in overall terms makes the marketing of microchips featuring these spray tips possible as a mass article. The proposed microstructure can be manufactured to advantage by hot embossing or by micro injection moulding. By means of such or similar processes, the fine structures required, such as are necessary for an ESI, and in particular a truncated cone with a tip diameter of some 10-100 xcexcm (micrometres) and a height of about 1 mm, can be manufactured to advantage.
By contrast with the prior art, in which at present the structuring of microchips is only possible on one side, the manufacturing process proposed according to the invention allows for an initial substrate to be structured on both sides; i.e. the formation on one side of the carrier of a channel structure with separation and delivery channels, and, at the same time, on the other side of the carrier the formation of a micro tip. The connection channel required between the channel structure and the spray tip can be created to advantage by laser drilling.
The channel structure can also be sealed or closed off to the outside by means of a cover plate, which is applied onto the carrier, following the manufacture of the micro tip. The connection channel between the channel structure and the spray tip can to advantage be formed as early as in the laser or injection mould, in particular in one work sequence with the manufacture of the micro tip and/or the channel structure. This allows the manufacturing costs to be further reduced.
In a further embodiment, an electrical connection is created for the spray tip by means of what is referred to as a xe2x80x9cliquid junctionxe2x80x9d. The electrical supply to the spray tip is effected to advantage in this case by means (via the connection channel) of the substances being fed to the spray tip. This embodiment accordingly does not require any additional electrical devices, for example in the form of a metallization layer or similar.
In order to prevent the possibility with the embodiment described of specimen molecules migrating into the connection channel (the channel for connecting the spray tip with the channel structure) instead of to the spray tip, it is to advantage for a pressure to be imposed at the connection channel of the liquid junction, increased by such a degree that the hydrodynamic flow created by this pressure is greater than or equal to the electro-osmotic flow (EOF). In addition to this, by specific adjustment of these pressure conditions, by the provision of an electrical connection for the spray tip, it is also possible in a specific manner for an additional liquid to be mixed into the substances which are to be examined, in order to optimise the ESI. For example, in this situation, organic acids may be added, such as formic acid or acetic acid, in order to improve the charging (proton building) of the substance specimen. As an alternative, or in addition, organic solvents such as methanol may be added, in order to optimise the spraying off or dispersion atomization.
The invention further relates to a device for handling a microchip according to the invention, in which provision is made for a flat formed carrier unit with hollow spaces arranged next to one another in the plane of the surface to accommodate the substances which are to be conducted to the microchip. The hollow spaces are in this case arranged in concordance with corresponding delivery apertures of the microchip in the said surface plane. In addition to this, the microchip can be connected to the carrier unit in a substance-conducting manner on the side which features the channel structure.
The proposed device accordingly allows for the use of standardised substance specimen holders (microtiter plates, known as xe2x80x9cwell platesxe2x80x9d), such as are already available on the market in a large number of automation robots and pipetting stations. Typically, the specimen transfer is effected from a well plate to a microfluidic microchip by pipetting up the liquids in each case or by the use of what is referred to as a transfer capillary, which is adhesively bonded into the microchip.
In particular, the device allows for the handling of the specimen and the specimen analysis to be combined in one function module, so that the specimen does not need to be additionally transferred from a well plate to the microchip. According to a preferred embodiment of the invention, the microfluidic microchip is designed in this context as a base piece of a well plate.
In addition, the proposed handling device allows for simplified automation, in particular of microchips with integrated spray tips. It is further advantageous for the micro tip, which forms the interface to the mass spectrometer, to be arranged on the underside of the microfluidic chip, so that the spray tip sprays downwards.
A further advantage of the device lies in the fact that the microchip can now be provided in the same geometric format as the dimensions of the well plate used in each case. Accordingly, during the handling of the specimen or the organisation of the specimen there is no longer any need to distinguish between the specimens themselves and the microchips. By the use of a standard frame for the arrangement of the delivery channels for the substances, it is possible to make use of conventional commercial specimen preparation devices for the off-line operation of the substance preparation.
Provision can further be made, instead of the use of an individual microchip, for a well plate rack to be provided, on which several microchips can be secured.
According to a further advantageous embodiment of the concept according to the invention, the microchip is designed longitudinally in relation to the plane of the surface, so that at least two microchips of longitudinal design of this nature can be arranged longitudinally next to one another in the plane of the surface, capable of being connected to the well plate in a substance-conducting manner. With this embodiment of the microchip, it is further of advantage for a separation channel to be arranged orientated in the longitudinal direction of the microchip, for the separation (analysis) of substances, in order thereby to maximise the (effective) length of the separation channel, and nevertheless avoid an excessively complex and elaborate channel arrangement. An arrangement of several such longitudinal microchips on a well plate further has the advantage that several different experiments can be carried out simultaneously on one single well plate. Thanks to the reduced number of well plates per microchip required with this arrangement, manufacturing costs are reduced on the one hand, and the time of the measuring cycles is reduced on the other.
The manufacturing process likewise proposed according to the invention, for a micro tip in question, features the special characteristic that the connection channel between the channel structure and the micro tip can be manufactured by a process which contains only a few stages. It is indeed possible with the prior art, by hot embossing or micro tip casting, to create micro tips with a tip diameter of about 15 to 100 xcexcm (micrometer), but with these small diameters it is not possible for the penetration aperture to be manufactured jointly (simultaneously) in one work stage, since the wall thickness would then only amount to a few micrometres. Accordingly, with the process according to the invention, a blind hole is first prepared by means of hot embossing, micro injection moulding, or an alternative manufacturing process is initially provided, of only about 30 xcexcm (micrometres) in front of the penetration point of the drill hole at the micro tip, so that only a thin membrane remains. It is then possible, with the aid of a laser or similar device, for the blind hole to be opened towards the tip.
To advantage, it is possible, at the spray tip, if appropriate even in one manufacturing process with the manufacture of the spray tip, for preference by laser ablation, for material located on the aperture diameter of the tip to be removed in order thereby to reduce the diameter of the tip even further.
The invention is described hereinafter on the basis of embodiments, whereby other objects, advantages, and features of the invention are derived from this description alone or in connection with the patent claims.